In manifold aspects of modern technology, particularly biotechnology, a need is present for methods of covalently binding molecules to solid matrices. While numerous methods of accomplishing these covalent bindings have been used or proposed, none have proven to be without technical difficulties and to be applicable in all situations. A most common method heretofore utilized for the binding of protein molecules to a solid matrix, for example, has been the reaction of proteins with polysaccharides such as Sepharose activated by cyanogen bromide [Axen et al. Nature (1967) 214 1302-1304]. This cyanogen bromide mediated binding however appears to produce an unstable covalent linkage [Tessen et al. (1973) FEBS Lett 23 56-58]. This procedure also introduces undesired positive charge into the matrix. The utilization of polyacrylamide matrices activated with glutaraldehyde to provide an aldehyde binding site for primary amino groups of proteins has been proposed [Weston et al. (1971) Biochem Biophys. Res. Comm. 45 1574-1580 and Ternynck et al. (1972) FEBS Lett 23 24-28] although later demonstrated not to be quantitatively dependable. Fiddler et al. [Anal. Biochem (1978) 86 716-724] demonstrated a method of derivatizing an aminoalkyl polyacrylamide gel (Aminoethyl Bio-Gel P-150) by treatment of the gel with glyceraldehyde followed by borohydride reduction of the resultant Schiff base to form a 1,2 dihydroxyalkyl Bio-Gel derivative. The dihydroxyalkyl Bio-Gel was then oxidized with aqueous metaperiodate to form an alkyl aldehyde Bio-Gel which was reacted with proteins bearing primary amino groups to form Schiff base linkages therewith. The Schiff base linkages were then reduced to stable secondary amine linkages by reduction with sodium cyanoborohydride.
Aminoethyl Bio-Gel P-150 is prepared by derivatizing polyacrylamide beads and is generally acknowledged by those skilled in the art to be positively charged due to the amine group substitutes. This positive charge lends itself to undesired antigenicity and may also permit non-specific ionic binding of charged molecules to the matrix. The chemical composition of the derivatized aminoethyl Bio-Gel P-150 matrix is incompletely defined. The Fiddler et al. reference is preferably limited to the use of pre-formed polyacrylamide beads with available amino groups; it is unusable for the de novo synthesis of various derivatized polyacrylamide physical forms.
Shainhoff [Biochem. Biophys. Res. Comm. (1980) 95 690-695] describes an acetaldehydic agarose derivative prepared by the coupling of glycidol and agarose followed by periodate oxidation of the alkyl 1,2-dihydroxy agarose to an aldehydic form. Binding of primary amine-bearing proteins to the aldehydic agarose was facilitated by sodium cyanoborohydride reduction of Schiff base linkages formed therebetween.
Agarose is acknowledged to be susceptible to attack by certain polysaccharide-hydrolyzing enzymes and is also potentially modifiable by treatment with inorganic periodates and moderately elevated pH levels. Agarose, being a polysaccharide, has significant antigenic potential and is also susceptible to undefined modifications by treatment with strong alkali such as 1M NaOH in the presence of NaBH.sub.4. The limited physical strength of agarose represents a limit on the potential physical uses of agarose derivatives.
In U.S. Pat. No. 4,180,308 and U.S. Pat. No. 4,401,372, both issued to Mancini et al., hydrogel contact lenses of hydrated terpolymers comprising dihydroxylalkylacrylate or methacrylate were disclosed. These monomers contain a potentially unstable ester group between the dihydroxy group and the matrix. U.S. Pat. No. 3,883,299, issued to Baumgarte et al. disclosed a textile dyeing process with sulfur dyes and reductones such as 2,3-dihydroxyacrylaldehyde. U.S. Pat. No. 4,154,747, issued to Epple et al., disclosed a process for producing 1-amino-8-nitro-4,5-dihydroxyanthroquinone, one step of which entailed the reductive use of 2,3-dihydroxyacryaldehyde. U.S. Pat. No. 4,390,683, issued to Yatsu et al., disclosed a stretched poly-1,3-phenylene terephthalate film optionally comprising 3-hydroxyphenyl-1,2-dihydroxyacrylate.
All of the procedures described above have one or more of the following potential disadvantages. (a) The linkage between the dihydroxy functional group and the matrix is chemically unstable, which can result in leakage or loss of ligand. (b) High levels of ionic charge are present in the matrix which can promote non-specific and unwanted interactions with other molecules and which can make the matrix antigenic. (c) The chemical composition and nature of all functional groups in derivatized matrices are unknown. This may preclude efficient chemical utilization of the matrix, may increase the non-specific binding of undesired molecules to these unknown side products and may prevent approval of the matrix for use in humans.